Characterization of gene expression regulation using D-RECS polymer by enzymatic reaction for an effective design of enzyme-responsive gene regulator

摘要

The development of targeted gene delivery systems has attracted much attention. Nevertheless, target cell-specific gene delivery has not been realized. However, if gene expression can be regulated with a gene carrier, this method could be a powerful tool for target specific gene therapy. We have previously reported some novel artificial gene-regulation systems responding to intracellular signaling enzymes by using graft-type polymers that contain substrate peptide (conjugate) for a target enzyme. The conjugate is able to regulate gene transcription in a cell-free system, in cultivated cells and also in in vivo system. We termed this concept 'D-RECS' which means Drug or gene delivery system responding to cellular signals. In the present study, in order to elucidate the mechanism of this gene expression regulation using the conjugate through enzymatic reactions, we characterized a high order structure of the conjugate/DNA complex and the ability of this conjugate to control gene expression using a Caspase-3 responsive system as a typical example. In addition, we compared these properties with those of a substrate peptide/DNA complex. As a result, we elucidated the importance of the polymer backbone for gene regulation. Because our conjugate contains a large amount of neutral polymer chain, wrapping of the DNA strand with a polymer chain acts as a suppressor for 'sliding' or 'jumping' of sequence-recognizing enzymes such as RNA polymerase or restriction enzymes so that the conjugate can efficiently suppress gene expression or site-specific cleavage. Furthermore, our conjugates formed a loosely packed complex with DNA. This behavior is essential for enzymatic regulation of gene transcription because the complex permits access of essential enzymes such as Caspase-3 but prohibits the access and/or sliding of RNA polymerase.